Interpretive Summary: Rice is the staple food for half the world’s population. While most rice is consumed as whole kernel cooked rice, the consumer's preference for cooked rice texture and other sensory properties differs among regions of the world. Rice is also used as an ingredient in a multitude of foods by food processing companies across the globe. These sensory and functional properties of rice are predominantly associated with its apparent amylose content (AAC). Thus, rice breeders around the world developing breeding lines with specific end-use properties often select for specific AAC. However, AAC and other processing related properties, such as starch paste viscosity, are traits known to be variably affected by environmental factors, such that the measured AAC of a variety grown in any one environment may not be the typical AAC that variety would have averaged over several years and locations. When genetic markers significantly associated with desirable traits are available, marker aided selection for traits can reduce or eliminate ambiguities that are introduced by environmental factors. Recent studies identified three functional single nucleotide polymorphisms (SNPs) in the Waxy gene and associated these SNPs strongly with AAC and pasting properties. These findings provide opportunities for the selection of desired end-use quality traits using molecular genetic technologies. The methods reported in the original identification of these SNPs and their associations are either low throughput genotyping methods or required special instrumentation, as well as skills not typically accessible in molecular breeding programs. We have developed three allele-specific markers to genotype three functional SNPs in the rice Waxy gene in a single PCR amplification. These assays are as simple as, and use the same instruments as, those for microsatellite markers. Therefore, these markers can be easily implemented by breeding programs using marker assisted selection technology for these important grain quality traits.

Technical Abstract:
Four Waxy haplotypes, which were identified previously as having different combinations of these three single-nucleotide polymorphisms (SNPs) of the Waxy gene, were highly correlated to the apparent amylose content and pasting properties, the important grain quality traits for predicting cooked rice texture and processing properties (Chen et al. 2008a and b). Three allele-specific markers were developed to genotype three functional SNPs in the rice (Oryza sativa L.) Waxy gene in a single PCR amplification. Each marker contains two allele-specific primers and one common primer. Each set of the allele-specific primers differed by one base at the 3'-end specifically discriminating SNP alleles and were labeled with different fluorescence probes. An additional mismatched base, the third base from the 3'-end, was inserted in some allele-specific primers to increase selectivity. The amplification step of the PCR thermal cycling program was set initially for 20x touch-down cycles having the annealing temperature of the first cycle approximately 6'C above the thermal melting temperature of all three primers at a touch-down rate of -0.3'C per cycle, and then followed with 25x regular thermal cycles having the annealing temperature at their thermal melting temperature. The allelic genotypes for each SNP were distinguished from each other by both their differential primer-allele fluorescences and their amplification product lengths. The simplicity of these assays suggests that these markers can be easily implemented by breeding programs using marker assisted selection technology for these important grain quality traits.